Effects of kallikrein gene transfer on penumbral microvascular proliferation and on regional cerebral blood flow following cerebral ischemia/reperfusion injury

BACKGROUND: Recent findings have demonstrated that the kallikrein-kinin system (KKS) participates in the pathological process of cerebral ischemia/reperfusion injury. Kallikrein gene transfer exhibits neural protective effects following cerebral infarction.OBJECTIVE: To observe the effects of kallikrein gene transfer on vascular proliferation in the peripheral infarct focus and on regional cerebral blood flow (rCBF) following cerebral ischemia/reperfusion injury. DESIGN, TIME AND SETTING: The completely randomized, controlled experiment was performed at the Lin Baixin Laboratory Center, the Second Affiliated Hospital of Sun Yat-sun University between September 2007 and April 2008.MATERIALS: pUCI9-HTK plasmid was constructed and maintained in the Laboratory for Neurology, the Second Afftliated Hospital of Sun Yat-sen University, China. Mouse anti-human kallikrein 1 monoclonal antibody was purchased from R&D Systems,USA.METHODS: Ninety healthy, male, Sprague Dawley rats were used. Middle cerebral artery occlusion (MCAO) was established in all rats to induce cerebral ischemia/reperfusion injury. Following MCAO establishment, all rats were randomly divided into three groups (n = 30): blank control, saline, and pAdCMV-HTK. The saline and pAdCMV-HTK groups were stereotactically micro-injected with 5 μ L of physiological saline or with pAdCMV-HTK Imultiplicity of infection (MOI)= 20], respectively, into the ischemic penumbra. In the blank control group, only sham injection was performed.MAIN OUTCOME MEASURES: At 12, 24, and 72 hours after treatment, cerebral infarction volume was measured by 2, 3, 5-triphenyltetrazolium chloride (TIC) staining. Exogenous HTK expression, as well as regional vascular endothelial growth factor (VEGF) expression, was detected by immunohistochemistry. rCBF was examined by 14C-iodoantipyrine micro tracing. In addition, neurological severity score (NSS) was performed. Higher scores indicated more severe neurological deficits.RESULTS: NSS results demonstrated that compared with the saline and the blank control groups, the pAdCMV-HTK group exhibited lower NSSs 24 hours after pAdCMV-HTK injection (P < 0.05). The NSSs were further decreased after 72 hours (P < 0.01). Cerebral infarction volume at 24 hours, and in particular at 72 hours after treatment, was significantly reduced in the pAdCMV-HTK group compared with the blank control and saline groups (P < 0.05). The rCBF in the area surrounding the infarction lesion was slightly decreased in all groups compared with the contralateral area. At 24 and 72 hours following treatment, the rCBF in the peripheral infarction lesion was significantly elevated in the pAdCMV-HTK group compared with the blank control and saline groups (P < 0.05). Immunohistochemistry results revealed that VEGF-positive cells were primarily found in the cortex and in some white matter surrounding the cerebral infarction lesion. In addition, the expression of VEGF in the pAdCMV-HTK group was significantly higher compared with that in the blank control and saline groups at 12, 24, and 72 hours following treatment (P < 0.05). CONCLUSION: Following cerebral ischemia/reperfusion, kallikrein gene transfer can promote vascular proliferation in the brain tissue surrounding the infarction lesion, improve rCBF, and reduce infarction volume, thereby exhibiting protective effects to attenuate neurological deficits.